Interferon is a glycoprotein whose synthesis in cells is principally induced by viruses or mitogens. Interferons are classified into three major species designated alpha-IFN (leukocyte), beta-IFN (fibroblast), and gamma-IFN (immune). Interferons possess potent antiviral, anticellular, immunoregulatory, and antitumor activities. Interferon treatment of cancer and viral infections in animals has met with some success. Recent evidence suggests that infections caused by other etiological agents may yield to gamma-IFN treatment. It has been reported that gamma-IFN strongly inhibited the development of the exoerythrocytic form of Plasmodium berghei in the liver cells of murine hosts. Human gamma-IFN also diminished the parasitemia in chimpanzees infected with sporozoites of the human malaria parasite, Plasmodium vivax. In addition, initial clinical tests with human gamma-IFN in the treatment of patients with lepromatous leprosy were encouraging enough to suggest further testing for therapeutic effects of gamma-IFN in non-viral infectious diseases. Gamma-IFN, therefore, is of great interest clinically as well as academically.
In general, recombinant deoxyribonucleic acid (DNA) techniques have now become well known. An extensive technical discussion embodying most commonly used recombinant DNA methodologies can be found in Molecular Cloning: A Laboratory Manual (1982) by Maniatis, Fritsch and Sambrook. Genes coding for various polypeptides may be cloned by incorporating a DNA fragment coding for the polypeptide in a recombinant DNA vehicle, e.g., bacterial or viral vectors, followed by transformation of a suitable host. Previously, this host was primarily Escherichia coli (E. coli) cells. However, with the development of eukaryotic viral vectors, it is now possible to reintroduce cloned genes into their natural environment, where mechanisms involved in control of gene expression and developmental regulations can be tested. A important application of eukaryotic vectors is the production of biologically active gene products suitable for pharmacological use.
Several methods are currently in use for delivering defined foreign DNA segments into eukaryotic cells. These include physical injection of DNA, fusion of DNA containing liposomes or erythrocytes with target cells, the direct application of naked DNA onto cells in the presence of calcium phosphate and marker DNA, protoplast fusion and electroporation.
Insertion of DNA into recipient cells has also been achieved by using viral particles in which a segment of the viral genome is covalently linked to defined nucleic acid segments. One such example is the Simian Virus 40 (SV40) vector system, which offers a rapid and efficient way to introduce foreign DNAs into permissive host cells. The system is limited by the size of DNA that can be accommodated within the virus particle. Moreover, since monkey cells are permissive for SV40 replication, infection by recombinant SV40 particles culminates in cell death. SV40 DNA has not yet been exploited as a cloning vector in nonpermissive rodent cells because (i) SV40 transformation is associated with integration of the viral genome, a process that may disrupt the integrity of the foreign DNA segment of interest; and (ii) there is no indication that the gene will be active at detectable levels from the low integrated copy numbers which are sufficient for the expression of the SV40-transforming gene. Other recombinant viral particles have also been used for specific applications in gene expression. These include retroviruses for potential in vivo utility in gene therapy, vaccinia virus for human and animal vaccination, and adenovirus for in vitro gene expression.
An example of a eukaryotic expressional vector which contains viral genetic components not incorporated within a virus particle is the bovine papillomavirus (BPV) DNA. The use of this vector was originally based on the observation that the viral genome persists as an extrachromosomal plasmid in transformed cells. In this form, the cloned gene is maintained in a uniform sequence environment of the BPV minichromosome, eliminating potential problems associated with the integration of the cloned DNA into inactive regions of the host chromosome. This property was fully exploited in the establishment of BPV shuttle vector capable of replicating in prokaryotic and eukaryotic cells. Such vectors are efficient in directing the regulated expression from inducible promoters and in expressing gene products destined for intra- and extracelular location.
The BPV shuttle vector system provides an efficient method to produce high amounts of a human protein in mammalian cells. However, it is limited in its use to rodent and bovine cells, a factor which may be a disadvantage in the expression of larger human proteins. Specifically, extensive post-translational modification (i.e., glycosylation) may be species specific. Consequently, the same protein produced in a heterologous host may be immunogenic in a human subject. Additionally, certain BPV plasmids will produce a transformed phenotype when transfected into rodent cells. Therefore, studies involved with differentiated cells are not feasible due to the dedifferentiating effect of these transforming vectors.
This invention describes the application of an Epstein-Barr virus (EBV) derived vector which can overcome certain limitations of BPV expression vectors, while retaining the advantages of extrachromosomal replication an the ability to direct expression of heterologous DNAs. Defined genetic elements of the EBV genome have been shown to support stable replication of recombinant plasmids. These include the cis acting origin of plasmid replication (oriP) and the trans acting Epstein-Barr nuclear antigen (EBNA-1) protein.
We have incorporated genetic regulatory elements to direct expression of heterologous genes from the EBV-derived vectors. These include, for example, the mouse metallothionein gene I promotor or SV40 early promotor, as well as enhancer sequences, such as those derived from the long terminal repeat (LTR) region of Moloney Murine Sarcoma Virus (M-MSV) or from human cytomegalovirus. DNA sequences from SV40 which direct splicing and specify polyadenylation of transcribed RNA have also been incorporated into the EBV-derived vectors. Such modifications direct the correct expression of heterologous genes, such as human gamma interferon or human Factor VIII, in human or monkey epithelial, fibroblast or lymphoid cells transfected with EBV-derived vector DNA. In addition, these vectors contain a dominant selectable marker, the gene for hygromycin phosphotransferase, the expression of which renders mammalian cells resistant to the antibiotic hygromycin-B (hyg.sup.r).
In cell lines having a large number of ribosomes, such as lymphocytes, it has been found that hygromycin-B is 3 to 10 times more toxic than G418, another aminoglycoside commonly used for selection of cells which have taken up DNA. Hygromycin-B may, therefore, be preferred to G418 in these cell types since it ensures a mor rapid selection of those cells harboring the DNA of interest. An additional advantage to using the EBV derived vectors is that the recipient host does not exhibit a transformed phenotype. Here we demonstrate the use of Epstein-Barr virus replicons as mammalian expression vectors.